Dental plaque is an example of a bacterial multi-species biofilm community. Development on a clean tooth surface is mediated by a tightly orchestrated and highly reproducible (from person- person) spatiotemporal succession of inter-bacterial adhesion events. During this process, pathogens can become part of the tooth-associated biofilm. If able to integrate, pathogens such as Porphyromonas gingivalis and Aggregatibacter (Actinobacillus) actinomycetemcomitans can rapidly divide and overcome the commensal biofilm population. This results in periodontal disease. The mechanism by which the pathogens can successfully integrate and expand in commensal biofilms is unknown. It is hypothesized in this exploratory proposal that inter-species bacterial communication contributes to the transition from a healthy dental plaque community to one that causes disease. Recent evidence suggests that autoinducer-2 (AI-2), an inter-species bacterial signal molecule, may mediate such a community change. AI-2, a product of the bacterial enzyme LuxS, is produced at picomolar concentrations by commensal bacteria and at higher concentration by pathogens. We propose an exploratory program to begin to elucidate the role of AI-2 based communication by growing wild-type and luxS mutants (that do not produce AI-2) in in-vitro flowcells. The contribution of AI-2 to (I) the spatiotemporal development of biofilms consisting of healthy bacterial species and (II) the integration of oral pathogens into these biofilms will be assessed. To achieve this, we have obtained wild-type and luxS mutants of commensal and pathogenic oral bacteria from research groups from around the world. We propose to grow wild-type and luxS mutants in single-, dual-, and tripartite-species in-vitro biofilms that are representative of conditions that are found in the human cavity. Human saliva will be used as the nutrient source and biofilms will be developed in flowcells for study by Confocal Laser Scanning Microscopy (CLSM). Fluorescently labeled antibodies, specific for each species, will be used in tandem with IMARIS(R) imaging software to discern spatiotemporal biofilm development. Thus a role for AI-2 in biofilm development and pathogen integration can be determined. Ultimately, this R21 exploratory program will significantly contribute to the understanding of the role of AI-2 in dental plaque biofilms and, if a role in biofilm colonization is discovered, facilitate in the development of novel therapeutic compounds to treat periodontal disease. PUBLIC HEALTH RELEVANCE: Dental plaque is composed of hundreds of species of bacteria living in close association, and some can cause periodontal disease. We propose that healthy and disease- causing species communicate with one-another by producing various small signal molecules that are collectively called autoinducer-2 (AI-2). Pathogenic species produce more AI-2 than the healthy species and an increased AI-2 concentration in dental plaque promotes the growth of pathogens at the expense of the healthy bacteria.